A research program concerned with the mechanism of metal ion regulated gene expression is described her. The project focuses on the transcriptional regulation of specific Cu-responsive genes. The aim of this is to characterize genes which are repressed by Cu, with the goal of identifying 1) copper responsive elements (CREs) which mediate its response to Cu, and 2) proteins which interact with these elements to serve as either inducers or repressors of transcription. The proposal is based on previous work which has shown that the accumulation of several proteins in the green alga Chlamydomonas reinhardtii is dependent on the concentration of Cu in the growth medium. One of these proteins, viz. cyt c-552, was shown to be controlled at the transcriptional level. In this application, the development of an in vitro transcription system, which is dependent on exogenous DNA and which can exhibit Cu-dependent initiation of transcription of the cyt c- 552 gene, is proposed. This system can be used to define CREs by deletion analysis of cloned genomic sequences. Alternatively, CREs can be identified by "reverse genetic" experiments where altered/deleted DNA sequences are introduced into the genome of host cells so that the effects of these deletions can be assayed in vivo. Once the CREs are identified, Cu-responsive trans-acting proteins which bind to these elements will be isolated by testing for specific DNA-protein interactions using gel retardation and footprint analyses. These assays can subsequently be used to purify the trans-acting proteins. This type of analysis will be extended to other cu-responsive genes; such as the gene encoding a 30kD soluble protein in C. reinhardtii which accumulates coordinately with cyt c-552 in response to Cu depletion. This previously identified 30 kD polypeptide will be purified so that DNa sequences encoding it can be identified and cloned; either by immunoscreening an expression library, or by the differential screening of cDNA libraries prepared from RNA isolated from Cu- deficient vs. Cu-supplemented cells. The identification of CREs in this gene will complement the cyt c-552 studies. The long term goal of the project is to elucidate the various steps involved in Cu-regulated gene expression--starting with the molecular recognition of Cu, the formation of macromolecular complexes, and resulting in the repression or expression of a regulated gene. This will be achieved by reconstruction of the system from the isolated components, and by genetic analysis of the regulatory pathway. protein-nucleic acid interactions are important events not only in transcription, but in the aspects of gene expression, including DNA replication, RNA processing and stability, and translational regulation. The recent identification of "Zn finger" domains in various transcription factors points to the key role of regulatory metalloproteins. In addition, this work will ultimately add to our understanding of metal binding, transport and metabolism and may result in information which could illuminate the causes of diseases associated with Cu metabolism (e.g. Menkes' and Wilson's diseases).